Hepatitis B is a widespread and potentially fatal viral disease. Although its etiological agent--i.e. the hepatitis B virus--has been isolated a long time ago from the blood of patients suffering of the disease, the development of a commercial hepatitis B vaccine has faced considerable drawback: hepatitis B virus is a 42 nm particle called the Dane particle consisting of (a) a core containing the viral genome bound to the core protein and containing the core antigen (HBcAg) which is not immunogenic and (b) an envelope containing the immunogenic surface antigen (HBsAg). HBsAg is a group of morphologically heterogeneous complex macromolecular structures; it contains proteins, carbohydrates, glycoproteins and lipids, the main constituents of which are phosphatidylcholine, cholesteryl ester, cholesterol and triglycerides. Infection with hepatitis B virus (HBV) leads not only to the production of Dane particles but also to overproduction of 22 nm particles and filaments containing the elements of the surface envelope. It is known that these 22 nm particles are about 1000-fold more immunogenic than the monomer HBsAg protein but the replication and expression of HBV has been hampered for a long time by the lack of an appropriate cell culture system to grow the virus in vitro. The lack of in vitro culture systems in which the virus can be efficiently propagated led to the development of searches towards either synthesis of HBsAg-like molecules or expression of HBsAg in alternative host systems by using recombinant DNA technique. For instance, expression of HBsAg has been reported from yeast cells transformed with DNA vectors bearing the gene for HBsAg and it is known that the HBsAg synthesized in yeast is assembled into particles having properties similar to the 22 nm particles secreted by human cells (P. VALENZUELA et al. Nature, 298; 347-350; 1982 and R. A. HITZEMANN et al. Nucl. Ac. Res. 11; 2745-2763; 1983). The production of HBsAg by recombinant technique is engineered cell cultures, for instance engineered yeast strains cultures is well known to the art (see for instance European Patent application publication No. 0 106 828; W. J. McALEER et al. in Nature 307; 178-180, 1984 and C. E. CARTY et al. in Abstract 030 of the annual meeting of the American Society for Microbiology 1984, 84th meeting, Mar. 4-9 1984).
Numerous articles and patents describing processes of extracting and purifying HBsAg from plasma, serum or other blood products or biological fluids or engineered cell cultures have also appeared.
The process of the invention is applicable to engineered yeast cells producing HBsAg.
The extraction and purification of HBsAg from an engineered yeast cell culture which has been grown to a satisfactory cell density generally requires 3 successive steps which are:
(1) removing HBsAg from the cell interior; PA0 (2) enriching the medium in HBsAg; PA0 (3) eliminating substantially all contaminants from the medium.
In most procedures, step (1) is performed by mechanical forces such as shearing forces (for example X-press or French press) or shaking the glass beads, eventually with addition of a detergent. The use of a non ionic detergent (Triton X-100) has been reported by K. MURRAY et al. in The EMBO J. 3; 645-650; 1984 and by R. A. HITZEMAN et al. loc. cit.
It has also been reported that incubation of HBsAg with detergents breaks down the particle. With that view
GB Pat. No. 2 093 039 discloses treatment of HBsAg with a non ionic detergent (Triton X-100) to form a polypeptide mixture which is introduced on top of an aqueous buffer solution containing a sucrose gradient and recovering a fraction substantially free from detergent and containing micelles of the polypeptide mixture;
EP Pat. No. 0 005 864 discloses contacting antigenic mass containing hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) of a particle size of at least 20 nm with a detergent (e.g. Tween 20 or 80) followed by treating the detergent containing antigenic composition with an aqueous aldehyde solution. The obtained product is a subparticulate mass having a particle size less than 20 nm especially less than 5 nm.
Japanese Patent application No. 53 104 724 (Derwent abstract 75324 A) discloses a process wherein HBsAg particles are heated in the presence of (1) a non ionic surfactant (i.e. polyoxyethylene alkylphenyl with 7-10 oxyethylene chains). (2) a protein-denaturating agent (i.e. urea or guanidine) and (3) optionally a reducing agent. The obtained product has a particle size comprised between 10 and 20 nm.
Japanese Patent application No. 50 160 420 (Derwent abstract 27420 A) discloses a process wherein HBsAg is treated with different surfactants to produce subunit products.
W. German Patent application No. 26 11 723 discloses a process wherein HBsAg spherical particles are obtained by warming the antigen in presence of a defatting surfactant.
U.S. Pat. No. 4,113,712 discloses HBsAg particles composed of single polypeptide subunits prepared by heating HBsAg in isotonic sodium chloride solution at about neutral pH containing a surfactant capable of delipidation.
U.S. Pat. No. 4,481,189 discloses a process wherein blood plasma or plasma derivative is sterilized by contact with a detergent (e.g. a non ionic detergent).
Steps (2) and (3) incorporate a variety of techniques, among which adsorption/desorption using colloidal silica, more particularly `AEROSIL` (a product sold by DEGUSSA, Francfort, FRG.)
Such technique is described in the following references:
W. J. M. DUIMEL et al. in Vox Sang. 23; 249-255; 1972;
J. PILLOT et al in J. Clin. Microbiol. 4; 205-207; 1976 and in Molec. Immunol. 21; 53-60; 1984.
F. BARIN et al. in Ann. Microbiol. (Inst. Pasteur) 129B; 87-100; 1978
which teach that at best only a fraction (i.e. about 60%) of the adsorbed HBsAg is recovered (the best results being obtained by elution with a low ionic strength buffer) unless some amount of deoxycholate be added to the eluant.
In another study (SIEBKE et al. in Acta Path. Microbiol. Scand. Section B; 80; 935-936; 1972), it has been pointed out that Australian antigen is partly eluted from Aerosil by 2% Tween 80 and, likewise, is poorly adsorbed in the presence of the detergent.